Molecular Genetics of Glutamate Responses. We hypothesized that glutamate receptors in the medial prefrontal cortex mediate activate dopamine neurons in the VTA that may facilitate plastic mechanisms, such as neurotrophin release and synthesis, to produce conditioned or habit-forming behaviors. As the next conceptual stage in this work, we thought it likely that genetic variation among glutamate and neurotrophin receptors would play an important role in modifying neuronal plasticity. It is also plausible that allelic variation in shared signal transduction pathways among glutamate and neurotrophin receptors may also play a role in mediating early responses (sensitization) or conditioned responses (addiction) to alcohol or other drugs. In order to capture as much sequence variant data at glutamate receptor genes, we surveyed the entire family of iontrophic glutamate receptors (IGRs). We used screening technologies, including denaturing high-performance liquid chromatography (dHPLC), DNA melting analysis (Lipsky et al., 2001), and direct sequencing for screening, and information mining of public (e.g. dbSNP, ENSEMBL) and private (i.e. Celera Discovery System) sequence databases to discover novel coding region and promoter regions single nucleotide polymorphisms (SNPs). A collection of 38 missense variants were identified by the informatics and resequencing approaches in several of these receptor genes including GRIN2B, GRIN3B, GRIA2, GRIA3, and GRIK1. This represents only a fraction of the total sequence variation across the IGR genes, but in fact these may constitute a large fraction of the functional polymorphisms at these genes. Thus, these polymorphisms are a starting point for understanding the role of IGRs in neurogenetic variation. To identify candidate functional SNPs we generated PCR amplified products from a panel of genomic DNAs composed of 480 unrelated individuals of diverse clinical and ethnic background. We screened the 5? flanking, untranslated, and coding regions. DNA sequence variants were confirmed by direct sequencing of PCR products and in some cases, by RFLP analysis. Of particular interest were eight SNPs in NR1 and NR2B subunit genes. Four of the variants resulted in an amino acid change (missense variants). Three are candidate promoter variants. All of the variants were previously unidentified. In particular the NR1 gene variant 8396G>A resulted in a cysteine to tyrosine substitution at position 744. When expressed in human embryonic kidney cells with a native NR2A subunit, this receptor variant demonstrated significant alterations in electrophysiological responses to glutamate and showed increased resistance to NMDA-mediated excitotoxcity. The three GRIN2B missense variants, Ser116Thr, Leu120Ile, and Thr275Ala are localized to the extracellualr ligand binding or modulatory domain of the NR2B subunit. Their frequencies range from approximately 3% to 10% in different populations. Significant functional changes such as these could result in functionally distinct forms of the NMDA receptor, in vivo. Molecular Genetics of Brain Derived Neurotrophic Factor (BDNF). A number of studies have indicated that prolonged, major depression disorder (MDD) is associated with a selective loss of hippocampal volume that persists long after the depression has resolved. Ionotropic glutamate receptors (IGR) subtypes, and apoptotic cell death pathways are very likely to be involved in this process. The induction of endogenous neuronal pathways, such as those increasing BDNF levels, can prevent neuronal loss. In addition, glucocorticoids, the adrenal steroids secreted during stress, may also play a contributing role in neuron loss. The interplay of IGR activation, particularly receptors of the NMDA subtype, neurotrophin release and synthesis are critical to maintaining neuronal viability. Genetic variation in IGR genes, neurotrophin genes, and stress pathways are likely to profoundly influence neuronal viability. BDNF interacts with a receptor tyrosine kinase (TrkB), allowing for neuronal survival and synaptic strengthening. There is growing interest in BDNF and the TrkB gene (NTRK2) as candidate genes for vulnerability to addictions and other psychiatric conditions, including anxiety and MDD. Although temporal and spatial patterns of BNDF expression during neurodevelopment of mice and rats have been mapped, little is known about transcriptional regulation of the human BDNF gene. Four promoters are known to initiate transcription of BDNF. The primary DNA sequences of these promoter regions are highly conserved between rats, mice, and humans and appear to be differentially transcribed in the brain, at least in the rat. The human BDNF gene is composed of five exons, four 5? untranslated exons (1-4) are clustered on a 21.8 kb region of chromosome 11. Each BDNF transcript is initiated separately within the 5? flanking region from each of these untranslated exons. The primary transcripts are differentially spliced to a single 3? coding region, exon 5, which encodes the entire sequence for the mature polypeptide, located approximately 42 kb downstream from this cluster of untranslated exons. Previous studies supported an association of a functional BDNF variant (Val66Met) with bipolar disorder. However, direct evidence that this misense variant is the contributing to the disorder is still uncertain. To better understand the complex regulation of BDNF, we used variant discovery approaches (above) to identify two sequence variants in BDNF promoter regions and two variants in 5? untranslated exons 1 and 4. The variants were previously unidentified. Allele frequencies are greater that 10% in different patient populations and other ethnically-defined populations. In addition, we produced reporter constructs and have obtained preliminary data in both neuronal cell lines and primary hippocampal neurons that indicate that each of the promoter variants reduce BDNF-specific mRNA expression. To determine the genetic variation within the BDNF region and how it contributes to previous linkage evidence (Vall66Met polymorphism, exon 5), we incorporated each of these functional variants into an linkage disequilibrium (LD) mapping study. We examined haplotypes and LD patterns among seven single nucleotide polymorphisms (SNPs) within a 73 kb genomic region that covered the entire BDNF gene. Initially, we determined the haplotype structure of a cluster of sites, including a functional missense variant in the 3? end of the gene, covering a physical distance of approximately 28 kb. Genotyping was performed using 5?-exonuclease assays in two populations: a Finnish population (N = 754) and a Southwest American Indian population (N = 505). There was no significant deviation from Hardy Weinberg Equilibrium in either population. On the basis of haplotype frequencies, the level of LD between each pair of sites was assessed using D? as a measure of allelic association. LD was high (0.99) across the 3? end of BDNF in each population indicating the region is contained in a single haplotype block. We have also discovered new sequence variants in the promoter regions of human NTRK2. Current work is centered on defining the role of each of the functional promoter variants in vitro using reporter gene constructs in cell lines and primary hippocampal neurons (above), electrophoretic mobility shift assays (EMSA), and chromatin immunopreciptation assays (ChIP). We are also using differential allele-specific mRNA assays to determine potential influences of promoter variants on BDNF mRNA levels human post-mortem brain tissue samples that have been genotyped for different markers across BDNF.